技术资料

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation,however,remain to be fully elucidated. Here,we demonstrate that Sox8,a member of the SRY-related HMG box transcription factor family,is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression,which is the hallmark of functionally mature M cells. Furthermore,genetic deletion of Sox8 causes a marked decrease in the number of mature M cells,resulting in reduced antigen uptake in Peyer's patches. Consequently,juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses. View Publication

Myeloid-derived suppressor cells (MDSCs) can subdue inflammation. In mice with acute graft-versus-host disease (GVHD),donor MDSC infusion enhances survival that is only partial and transient because of MDSC inflammasome activation early posttransfer,resulting in differentiation and loss of suppressor function. Here we demonstrate that conditioning regimen-induced adenosine triphosphate (ATP) release is a primary driver of MDSC dysfunction through ATP receptor (P2x7R) engagement and NLR pyrin family domain 3 (NLRP3) inflammasome activation. P2x7R or NLRP3 knockout (KO) donor MDSCs provided significantly higher survival than wild-type (WT) MDSCs. Although in vivo pharmacologic targeting of NLRP3 or P2x7R promoted recipient survival,indicating in vivo biologic effects,no synergistic survival advantage was seen when combined with MDSCs. Because activated inflammasomes release mature interleukin-1$\beta$ (IL-1$\beta$),we expected that IL-1$\beta$ KO donor MDSCs would be superior in subverting GVHD,but such MDSCs proved inferior relative to WT. IL-1$\beta$ release and IL-1 receptor expression was required for optimal MDSC function,and exogenous IL-1$\beta$ added to suppression assays that included MDSCs increased suppressor potency. These data indicate that prolonged systemic NLRP3 inflammasome inhibition and decreased IL-1$\beta$ could diminish survival in GVHD. However,loss of inflammasome activation and IL-1$\beta$ release restricted to MDSCs rather than systemic inhibition allowed non-MDSC IL-1$\beta$ signaling,improving survival. Extracellular ATP catalysis with peritransplant apyrase administered into the peritoneum,the ATP release site,synergized with WT MDSCs,as did regulatory T-cell infusion,which we showed reduced but did not eliminate MDSC inflammasome activation,as assessed with a novel inflammasome reporter strain. These findings will inform future clinical using MDSCs to decrease alloresponses in inflammatory environments. View Publication

BACKGROUND Vitamin D deficiency is associated with intestinal barrier dysfunction,which contributes to pathogenesis of acute intestinal injury in children. We aim to investigate the effects of vitamin D on intestinal injury in intestinal epithelial cells and organoids. METHODS Lipopolysaccharide (LPS) was used to induce injury in intestinal epithelial cells (IEC-18) and organoids,and the effect of vitamin D was assessed. Cell viability was measured and inflammation cytokines TNF$\alpha$ and IL-8 were quantified. FITC-dextran 4 kDa (FD4) permeability was measured using Transwell while tight junction markers were assessed by immunofluorescence staining in IEC-18 and intestinal organoids. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS IEC-18 viability was decreased by LPS treatment,but was prevented by vitamin D. The upregulation of inflammation was inhibited by vitamin D,which also decreased epithelium permeability. Vitamin D restored tight junction ZO-1 and claudin 2. In addition,vitamin D decreased TNF$\alpha$ expression and prevented the disruption of ZO-1 in injured organoids. CONCLUSIONS Vitamin D rescued epithelial barrier function by improving permeability and restoring tight junctions,leading to decrease inflammation. This study confirms the protective effects of vitamin D,which could be used as a treatment strategy for infants at risk of developing intestinal injury. View Publication

Glucocorticoids (GCs) play an important role in controlling acute graft-versus-host disease (aGvHD),a frequent complication of allogeneic hematopoietic stem cell transplantation. The anti-inflammatory activity of GCs is mainly ascribed to the modulation of T cells and macrophages,for which reason a genetically induced GC resistance of either of these cell types causes aggravated aGvHD. Since only a few genes are currently known that are differentially regulated under these conditions,we analyzed the expression of 54 candidate genes in the inflamed small intestine of mice suffering from aGvHD when either allogeneic T cells or host myeloid cells were GC resistant using a microfluidic dynamic array platform for high-throughput quantitative PCR. The majority of genes categorized as cytokines (e.g. Il2,Il6),chemokines (e.g. Ccl2,Cxcl1),cell surface receptors (e.g. Fasl,Ctla4) and intracellular molecules (e.g. Dusp1,Arg1) were upregulated in mice transplanted with GC resistant allogeneic T cells. Moreover,the expression of several genes linked to energy metabolism (e.g. Glut1) was altered. Surprisingly,mice harboring GC resistant myeloid cells showed almost no changes in gene expression despite their fatal disease course after aGvHD induction. To identify additional genes in the inflamed small intestine that were affected by a GC resistance of allogeneic T cells,we performed an RNAseq analysis,which uncovered more than 500 differentially expressed transcripts (e.g. Cxcr6,Glut3,Otc,Aoc1,Il1r1,Sphk1) that were enriched for biological processes associated with inflammation and tissue disassembly. The changes in gene expression could be confirmed during full-blown disease but hardly any of them in the preclinical phase using high-throughput quantitative PCR. Further analysis of some of these genes revealed a highly selective expression pattern in T cells,intestinal epithelial cells and macrophages,which correlated with their regulation during disease progression. Collectively,we identified an altered gene expression profile caused by GC resistance of transplanted allogeneic T cells,which could help to define new targets for aGvHD therapy. View Publication

Autoimmune diseases are a physiological state wherein immune responses are directed against and damage the body's own tissues. Cytokines secreted by infiltrated inflammatory cells contribute to the pathogenesis of autoimmune diseases. TIPE2,one of the four family members of Tumor necrosis factor-$\alpha$ induced protein-8 (TNFAIP8),is a negative regulator of innate and adaptive immunity and plays essential roles in the maintenance of immune tolerance. However,studies on the role of TIPE2 during the development of autoimmune diseases have generated contradictory results. In the current study,we sought to determine the role of TIPE2 during the development of IMQ-induced psoriasis and Experimental Autoimmune Uveitis (EAU) in mice. Our study revealed that,while TIPE2-deficiency alleviates psoriasis,it exacerbates the development of EAU. Further studies demonstrated that,although TIPE2-deficient T cells produced more IL-17A,they do not migrate efficiently to the local inflammatory site,i.e.,the skin. This in turn led to the decreased IL-17A production in the skin and consequently reduced the severity of psoriasis in TIPE2-deficient mice. However,although TIPE2-deficient T cells still produced more IL-17A in EAU model,they migrate into the inflamed eye as efficient as TIPE2-sufficient T cells,and consequently exacerbates the development of EAU in TIPE2-deficient mice. Taken together,these results indicate that TIPE2 may either promote or suppress autoimmunity depending on the specific inflammatory microenvironment in different types of autoimmune diseases. View Publication

BACKGROUND MiR-199a-3p (miR-199a) can enhance the chemosensitivity of hepatocellular carcinoma (HCC). Because of the easy degradation of miRNA by direct infusion,effective vehicle-mediated delivery of miR-199a may represent a new strategy for improving HCC chemotherapy. Considering mesenchymal stem cell (MSC)-derived exosomes as promising natural nanovectors for drug and molecule delivery,we aimed to determine whether exosomes from adipose tissue-derived MSCs (AMSCs) could be used to deliver miR-199a and improve HCC chemosensitivity. METHODS MiR-199a-modified AMSCs (AMSC-199a) were constructed by miR-199a lentivirus infection and puromycin selection. MiR-199-modified exosomes (AMSC-Exo-199a) were isolated from the supernatant of AMSC-199a and were assessed by transmission electron microscopy,nanoparticle tracking analysis,and flow cytometry analysis. The expression levels of miR-199a in HCC samples,AMSCs,exosomes,and HCC cells were quantified by real-time PCR. The effects of AMSC-Exo-199a on HCC chemosensitivity were determined by cell proliferation and apoptosis assays and by i.v. injection into orthotopic HCC mouse models with doxorubicin treatment. MTOR,p-4EBP1 and p-70S6K levels in HCC cells and tissues were quantified by Western blot. RESULTS AMSC-Exo-199a had the classic characteristics of exosomes and could effectively mediate miR-199a delivery to HCC cells. Additionally,AMSC-Exo-199a significantly sensitized HCC cells to doxorubicin by targeting mTOR and subsequently inhibiting the mTOR pathway. Moreover,i.v.-injected AMSC-Exo-199a could distribute to tumor tissue and markedly increased the effect of Dox against HCC in vivo. CONCLUSIONS AMSC-Exo-199a can be an effective vehicle for miR-199a delivery,and they effectively sensitized HCC to chemotherapeutic agents by targeting mTOR pathway. AMSC-Exo-199a administration may provide a new strategy for improving HCC chemosensitivity. View Publication

Peripheral serotonin (5-hydroxytryptamine: 5-HT) synthesized in the intestine by enterochromaffin cells (ECs),plays an important role in the regulation of peristaltic of the gut,epithelial secretion and promotes the development and maintenance of the enteric neurons. Recent studies showed that the indigenous gut microbiota modulates 5-HT signalling and that ECs use sensory receptors to detect dietary and microbiota-derived signals from the lumen to subsequently transduce the information to the nervous system. We hypothesized that Clostridium ramosum by increasing gut 5-HT availability consequently contributes to high-fat diet-induced obesity. Using germ-free mice and mice monoassociated with C. ramosum,intestinal cell lines and mouse organoids,we demonstrated that bacterial cell components stimulate host 5-HT secretion and program the differentiation of colonic intestinal stem progenitors toward the secretory 5-HT-producing lineage. An elevated 5-HT level regulates the expression of major proteins involved in intestinal fatty acid absorption in vitro,suggesting that the presence of C. ramosum in the gut promotes 5-HT secretion and thereby could facilitates intestinal lipid absorption and the development of obesity. View Publication

Sustained,indolent immune injury of the vasculature of a heart transplant limits long-term graft and recipient survival. This injury is mitigated by a poorly characterized,maladaptive repair response. Vascular endothelial cells respond to proangiogenic cues in the embryo by differentiation to specialized phenotypes,associated with expression of apelin. In the adult,the role of developmental proangiogenic cues in repair of the established vasculature is largely unknown. We found that human and minor histocompatibility-mismatched donor mouse heart allografts with alloimmune-mediated vasculopathy upregulated expression of apelin in arteries and myocardial microvessels. In vivo,loss of donor heart expression of apelin facilitated graft immune cell infiltration,blunted vascular repair,and worsened occlusive vasculopathy in mice. In vitro,an apelin receptor agonist analog elicited endothelial nitric oxide synthase activation to promote endothelial monolayer wound repair and reduce immune cell adhesion. Thus,apelin acted as an autocrine growth cue to sustain vascular repair and mitigate the effects of immune injury. Treatment with an apelin receptor agonist after vasculopathy was established markedly reduced progression of arterial occlusion in mice. Together,these initial data identify proangiogenic apelin as a key mediator of coronary vascular repair and a pharmacotherapeutic target for immune-mediated injury of the coronary vasculature. View Publication

Products derived from bacterial members of the gut microbiota evoke immune signalling pathways of the host that promote immunity and barrier function in the intestine. How immune reactions to enteric viruses support intestinal homeostasis is unknown. We recently demonstrated that infection by murine norovirus (MNV) reverses intestinal abnormalities following depletion of bacteria,indicating that an intestinal animal virus can provide cues to the host that are typically attributed to the microbiota. Here,we elucidate mechanisms by which MNV evokes protective responses from the host. We identify an important role for the viral protein NS1/2 in establishing local replication and a type I interferon (IFN-I) response in the colon. We further show that IFN-I acts on intestinal epithelial cells to increase the proportion of CCR2-dependent macrophages and interleukin (IL)-22-producing innate lymphoid cells,which in turn promote pSTAT3 signalling in intestinal epithelial cells and protection from intestinal injury. In addition,we demonstrate that MNV provides a striking IL-22-dependent protection against early-life lethal infection by Citrobacter rodentium. These findings demonstrate novel ways in which a viral member of the microbiota fortifies the intestinal barrier during chemical injury and infectious challenges. View Publication

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously,iPSCs from the same patient were differentiated into ECs. On day 34,the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54,and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible. View Publication

Lambda interferons (IFN-$\lambda$s) are a major component of the innate immune defense to viruses,bacteria,and fungi. In human liver,IFN-$\lambda$ not only drives antiviral responses,but also promotes inflammation and fibrosis in viral and non-viral diseases. Here we demonstrate that macrophages are primary responders to IFN-$\lambda$,uniquely positioned to bridge the gap between IFN-$\lambda$ producing cells and lymphocyte populations that are not intrinsically responsive to IFN-$\lambda$. While CD14+ monocytes do not express the IFN-$\lambda$ receptor,IFNLR1,sensitivity is quickly gained upon differentiation to macrophages in vitro. IFN-$\lambda$ stimulates macrophage cytotoxicity and phagocytosis as well as the secretion of pro-inflammatory cytokines and interferon stimulated genes that mediate immune cell chemotaxis and effector functions. In particular,IFN-$\lambda$ induced CCR5 and CXCR3 chemokines,stimulating T and NK cell migration,as well as subsequent NK cell cytotoxicity. Using immunofluorescence and cell sorting techniques,we confirmed that human liver macrophages expressing CD14 and CD68 are highly responsive to IFN-$\lambda$ ex vivo. Together,these data highlight a novel role for macrophages in shaping IFN-$\lambda$ dependent immune responses both directly through pro-inflammatory activity and indirectly by recruiting and activating IFN-$\lambda$ unresponsive lymphocytes. View Publication

Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication